This lab studies the effectiveness of alcohol's properties to kill bacteria. More specifically, it tests the effectiveness of solutions killing bacteria and germs with varied percent alcohol levels. This lab also compares the difference between pure ethanol alcohol with the type of alcohol mixed in a solution found in many store bought hand sanitizers. Depending on the results of testing alcohol's effectiveness of killing bacteria, the lab will futher test the effectiveness of surfactant allantoin, benzalkonium chloride (SAB), another agent used to kill bacteria.
Hypothesis:
Solutions with 60% or more alcohol percentage will kill a sufficient amount of bacteria, where as percent levels below that may not pass health standards whether its pure ethanol alcohol or alcohol found in store bought hand sanitizers.
1. Attain ten 200mL beakers, filling two with chicken broth, an environment to feed bacteria, as a control. The other eight beakers will be used to test the effectiveness of alcohol on bacteria with varied percent-alcohool levels.The first beaker will be 20% alcohol, 80% chicken broth. The second beaker 40% alcohol, 80% chicken broth. The third beaker will be 60% alcohol, 40% chicken broth, and the last beaker wil be 80% alcohol, 20% chicken broth. Each will be filled with 200mL of solution.
2.In each test tube insert bacteria in each of the beakers.
Wait for approximately two days and examine the beakers, taking note of how each of the control beakers look. Compare and contrast the colors of each beaker- which ones were cloudy- or which ones appeared to kill the most bacteria. Record data and take pictures of the results.
3. Attain at least three different types of hand sanitizer with varying amounts of alcohol percentages. Attain five 100mL beakers, filling the first two with 75mL of chicken broth. Fill the next three with 75mL of the hand sanitizer solutions. Repeat step two of the experiement.
4. If the past two tests on pure ethanol alcohol and store bought hand santiziers provide sufficient and accurate data, continue on to test the effectiveness of surfactant allatonin, benzalkonium chloride (SAB). Use the strongest alcohol percent solution from the pure alcohol test and the store bought alcohol test and create a solution of the same percentage of SAB.
5. Repeat steps to test with bacteria and record results. Make a table comparing the effectiveness of alcohol found in store bought hand sanitizers with the effectiveness of pure alcohol with the effectiveness of SAB solution.
Apparatus and Chemicals Needed
-Ten 200mL beakers
-Five 100mL beakers
-Chicken Broth
-Bacteria
-Ethyl Alcohol
-Three types of store bought hand sanitizers with varying percent alcohol levels, one under 60%, above 60%, and at 60%
-Allatonin benzalkonium chloride (SAB)- permitting the first two parts of the experiement appear accurate
Safety information: Chemicals/Reaction
Safety information:
- Wear goggles and gloves to protect yourself from potential harm
Ethyl Alcohol:
- Clear/colorless liquid
- Strong odor
- Boiling point of 80 degrees Celsius
- Irritates skin and eyes if contact is made
Precautions to take with Ethyl Alcohol: Keep away from heat, sparks and flame. Keep container closed. Use with adequate ventilation. Wash thoroughly after handling. Avoid contact with eyes, skin and clothing. Avoid breathing vapor. Benzalkonium Chloride: -Aromatic -Solid -White to Yellowish -Soluble in cold and hot water as well as alcohol Precautions to take with Benzalkonium Chloride -Keep away from eyes and direct contact with skin -Flammable in excess heat -Avoid Inhaling Allantoin:
-Solid
-Odorless
-Tasteless
Precautions to take with Allantoin:
-Wash if it touches skin
-Do not inhale
Other Information
Emily Nordquist Ms. Seibel Hunt Chemistry- 8 21 February 2012 Research on Efficacy of Hand SanitizerProperties of Ethanol on Bacteria: Ethanol is a dehydrator. This means that it ruptures bacteria cells by damaging the cell membrane and denaturalizing the cell until it lyses. This is different from hand soap which simply takes way the outer layer of oil that lies on a hand and which stops other bacteria that is already within the skin from rising to the surface. This is seen as not as effective as hand sanitizers which use ethanol. It has been tested that 70% percent ethanol concentrate will kill off 99.9% of germs or bacteria. One thing that causes hand sanitizers to be more effective than hand soap is the contact time it has with the skin. Negative Properties of Ethanol as Disinfectants: If the concentrate is under 60% it will not be as effective and maybe even just cause the bacteria to spread around more. Since ethanol kills germs by dehydration, it can sometimes be selective and therefore does not kill certain species of bacteria that actually resist ethanol (sterilizing selectivity). For example, bacterial spores are not typically affected by ethanol. Because of its selectivity, ethanol tends to work better on a pure mixture versus a mixed culture. Other Substitutes for Ethanol Bleach tends to work most effectively when killing bacteria. In a lab, it was shown that it worked more effectively on both the mixed and pure culture on E.Coli. It also appeared to act more quickly and be less selective. Hand soaps use an agent called triclosan, which actually proved to be very ineffective and had hardly any effect on killing E.Coli. Therefore, hand sanitizers might actually be a safer bet when washing hands because of the longer amount of contact time and the more direct dehydration of bacteria cells. Works Cited "Comparison study on disinfectant efficiency of." University of Kansas . University of Kansas, n.d. Web. 20 Feb. 2012. <www.cte.ku.edu/gallery/visibleknowledge/sturm/files/Student%20Work%20Documents/final%20Lab%20Project/group_b_report.pdf>.
Abstract: Efficacy of ethanol against E. Coli. Saif Ahmed and Emily Nordquist. The purpose of this lab was to test how effective ethanol is against bacteria, specifically Escherichia Coli. To test how effective ethanol is against E. Coli, solutions of chicken broth were mixed in different percentages of dilution. There were ten 200 mL beakers of solution total; two were 100% chicken broth, two were 80% chicken broth and 20% ethanol, two were 60% chicken broth and 40% ethanol, two were 40% chicken broth and 60% ethanol, and the final two were 20% chicken broth and 80% ethanol. After all of the solutions were made and after bacteria colonies were made in ten different agar plates, the two were mixed. To mix them, 1 mL of each solution was put into their respective agar plates. After a night of settling, results showed that there was no bacteria in any of the agar plates except for the two that did not have any ethanol. However, the lab did produce some unexpected results: at the bottom of almost every single solution mixture was an unknown white substance. However, in beakers 3 and 4 which had ethanol levels of 20%, the white substance was not present. According to biofuelguide.net, biodiesel is produced from alcohol (ethanol) and vegetable or animal oils/fats when the alcohol level is about 20%, which was the level of alcohol in beakers 3 and 4.
Keywords: Ethanol, chicken broth, E. Coli, agar plate, Petri dish,
Introduction: Several hand sanitizer brands like Purell and Germ-X advertise for killing 99.99% of germs. The active ingredient in each of these products is ethyl alcohol and each one contains a certain percentage of ethanol. Hand sanitizers have increasingly been placed in schools throughout the United States to prevent the spread of sickness and germs amongst the student body. However, questions remain as to how effective the some shelf brand hand sanitizers are that have less that 60% concentration of ethanol (Franklin 2006). However, sources such as The University of Chicago Press’s Infection Control and Hospital Epidemiology’s article state that the percentage of ethanol must be at or above 50% for the product to be effective in killing viruses and bacteria (Fendler, Groziak 2002). Therefore, the question remains as to which percentage of ethanol truly kills off bacteria and what ethanol concentration should be required in hand sanitizers. The University of Chicago tested the efficacy of Purell (62% ethanol) by applying different viruses to the product and using the process of plating to determine the efficacy of the hand sanitizer in killing bacteria. This lab was modeled after The University of Chicago’s test and determines the efficacy of varied ethanol percentages in killing E.Coli. By testing percentages of ethanol varying from 0% to 80% concentration combined with chicken broth, or a nutrient broth for the E.Coli, this lab observed the actual percentage of ethanol needed to kill off E.Coli, a very common bacteria. After letting the E.Coli grow in a separate nutrient broth for two days to feed and grow, it was then mixed with the ethanol-chicken broth solutions. The University of Chicago noted that the viruses were killed in approximately thirty seconds (Fendler, Groziak 2002). Therefore, the ethanol was expected to kill the E.Coli relatively quickly. Samples of each solution were taken and placed in an agar, a gelatinous substance, to begin the process of plating and counting. Plates contain nutrient in the agar for bacteria to feed on and reveal single colonies of bacteria and can be “counted.” This process of counting showed how many colonies were left over from the effects of each solution of ethanol and chicken broth. Two controls were also used to compare how much bacteria was present in the room since the lab was not completely free of bacteria, or sterilized. The ineffectiveness or effectiveness of the different solutions of ethanol and chicken broth can be compared to the different percentages of ethanol in different hand sanitizers. For example, one type of Purell is 70% ethanol while Target Brand and Germ-X are closer to 62-65% ethanol. Therefore, the 80% ethanol solution is more comparable to Purell whereas the 60% ethanol is more comparable to Target Brand and Germ-X sanitizers.
Methods:
The first step of this lab is to measure out chicken broth and ethanol into different levels of concentration. With a total of ten beakers, 2 will be filled with 200 mL of chicken brother, 2 will be filled with 160 mL chicken broth and 40 mL of ethanol, 2 will be filled with 120 mL of chicken broth and 80 mL of ethanol, and so on. After all the different mixtures are made, 10 bacteria colonies will be made in Petri dishes using natural agar. After the colonies are made, the number of bacteria will need to be counted. Once all the bacteria in each dish is counted, mix the solutions with the bacteria colonies and let sit for a while. After at least a day of letting sit, return to the Petri dishes and count the number of bacteria in each solution. This will tell you which percentage of ethanol concentration is the most effective.
Results:
Table 1: Observations of solutions of Chicken Broth and Ethanol (No E. Coli)
Ethanol percentages and beaker numbers
Observations
0% (1 & 2)
Saturated white stuff at the bottom
20% (3 & 4)
Very little to no stuff at the bottom
40% (5 & 6)
Clumpy white stuff at the bottom
60% (7& 8)
Clumpier white stuff at the bottom
80% (9 & 10)
Even clumpier white stuff at the bottom
Table 2: Chicken Broth + E. Coli + Ethanol (dishes 5-10 have too much solution)
Ethanol percentages and dish numbers
Observations
0% (1 & 2)
Bacteria colonies on both dishes, small white circle colonies spread all around
20% (3 & 4)
Very little to no evidence of bacteria colonies
40% (5 & 6)
Very little to no evidence of bacteria colonies
60% (7& 8)
Very little to no evidence of bacteria colonies
80% (9 & 10)
Very little to no evidence of bacteria colonies
The results from our experiment show that when the ethanol percentage is above 20%, E. Coli is eliminated.
Figure 1: Petri dish #1 – Little to no bacterial evidence
Figure 2: Petri dish #4 – bacteria colonies scattered across the dish
Discussion
The lab yielded unexpected results and refuted the hypothesis that the E.Coli would survive under a 60% concentration of ethanol alcohol. When referring to figures 13-23, it remains clear that bacteria were only present in Petri-dishes 1 & 2. In other words, bacteria only grew and survived in the controls which contained 100% chicken broth and 0% bacteria. This proves that the chicken broth did work as a sufficient nutrient broth for E.Coli. However, this also shows that E.Coli did not survive in 20% or higher concentrations of ethanol. This, however, does not allow one to conclude that 20% ethanol concentrations kills all bacteria due to the fact an unexpected reaction between the chicken broth ethanol took place and formed a white substance (Figures 18-19). This white substance appeared in all beakers besides beakers 3 & 4 (20% ethanol concentrations) and appeared more solid in beakers 5-10 and more liquid like in beakers 1 & 2. This provides evidence that perhaps a type of fat or oil settled to the bottom of beakers 1 & 2 due to natural separation, whereas in beakers 3-10 a reaction occurred between a type of oil or lipid with the ethanol. The reason that there was no substance present in beakers 3-4 is perhaps because the ethanol and chicken broth had fully reacted with each other at a 4-1 ratio (80% chicken broth, 20% ethanol) and no layers were able to form, where as beakers 5-10 did not have a perfect reaction ratio and therefore began to create layers.
This lab indicates that E.Coli grown in chicken broth will be killed in ethanol concentrations above 20%. It also shows that a more accurate test to determine at what concentration of ethanol E.Coli will be killed would be to test ethanol percentages between 0% and 20%. This lab also suggests that research should be done about the specific ingredients, primarily the oils and lipids in chicken broth and how they react with ethanol alcohol. Most likely, the ingredient will have a 4:1 reaction ratio due to the fact that no white substance was found in beakers 3 & 4. Another question to look at would be how well does chicken broth work to grow E.Coli versus a standard nutrient broth. One potential error that could have effect the lab results was also that each solution perhaps did not get an equal amount of E.Coli due to the fact the E.Coli was mixed in a liquid solution and did not guarantee equal distribution. The white substance formed could have also had an effect on killing E.Coli. Comparing these results to the varying percentages of ethanol in hand sanitizers; it appears as though when E.Coli is grown in circumstances similar to the chicken broth nutrient solution, it will be killed with 20% ethanol hand sanitizers. However, this lab does not guarantee that other types of bacteria will also follow these standards or guarantee that E.Coli not grown under circumstances like the chicken broth will follow these given standards.
Hand Sanitizers vs. Soaps
1) Franklin, Deborah. "Hand Sanitizers, Good or Bad? - New York Times." NY Times Advertisement. N.p., n.d. Web. 5 Jan. 2012.
2) Fendler, E., and P. Groziak. "Efficacy of Alcohol based Hand Sanitizers against Fungi and Viruses." JSTOR. Version Vol. 23 No. 2. Chicago Journals, n.d. Web. 6 Jan. 2012.
3) Fletcher, Madilyn. " The Effects of Methanol, Ethanol, Propanol and Butanol on Bacterial Attachment to Surfaces." Journal of General Microbiology. Department of Environmental Sciences, University of Warwick, 2 Aug. 1982. Web. 21 Feb. 2012.
4) "Comparison study on disinfectant efficiency of." University of Kansas . University of Kansas, n.d. Web. 20 Feb. 2012.
2.In each test tube insert bacteria in each of the beakers.
Wait for approximately two days and examine the beakers, taking note of how each of the control beakers look. Compare and contrast the colors of each beaker- which ones were cloudy- or which ones appeared to kill the most bacteria. Record data and take pictures of the results.
3. Attain at least three different types of hand sanitizer with varying amounts of alcohol percentages. Attain five 100mL beakers, filling the first two with 75mL of chicken broth. Fill the next three with 75mL of the hand sanitizer solutions. Repeat step two of the experiement.
4. If the past two tests on pure ethanol alcohol and store bought hand santiziers provide sufficient and accurate data, continue on to test the effectiveness of surfactant allatonin, benzalkonium chloride (SAB). Use the strongest alcohol percent solution from the pure alcohol test and the store bought alcohol test and create a solution of the same percentage of SAB.
5. Repeat steps to test with bacteria and record results. Make a table comparing the effectiveness of alcohol found in store bought hand sanitizers with the effectiveness of pure alcohol with the effectiveness of SAB solution.
-Five 100mL beakers
-Chicken Broth
-Bacteria
-Ethyl Alcohol
-Three types of store bought hand sanitizers with varying percent alcohol levels, one under 60%, above 60%, and at 60%
-Allatonin benzalkonium chloride (SAB)- permitting the first two parts of the experiement appear accurate
- Wear goggles and gloves to protect yourself from potential harm
Ethyl Alcohol:
- Clear/colorless liquid
- Strong odor
- Boiling point of 80 degrees Celsius
- Irritates skin and eyes if contact is made
Precautions to take with Ethyl Alcohol:
Keep away from heat, sparks and flame.
Keep container closed.
Use with adequate ventilation.
Wash thoroughly after handling.
Avoid contact with eyes, skin and clothing.
Avoid breathing vapor.
Benzalkonium Chloride:
-Aromatic
-Solid
-White to Yellowish
-Soluble in cold and hot water as well as alcohol
Precautions to take with Benzalkonium Chloride
-Keep away from eyes and direct contact with skin
-Flammable in excess heat
-Avoid Inhaling
Allantoin:
-Solid
-Odorless
-Tasteless
Precautions to take with Allantoin:
-Wash if it touches skin
-Do not inhale
Ms. Seibel Hunt
Chemistry- 8
21 February 2012
Research on Efficacy of Hand SanitizerProperties of Ethanol on Bacteria:
Ethanol is a dehydrator. This means that it ruptures bacteria cells by damaging the cell membrane and denaturalizing the cell until it lyses. This is different from hand soap which simply takes way the outer layer of oil that lies on a hand and which stops other bacteria that is already within the skin from rising to the surface. This is seen as not as effective as hand sanitizers which use ethanol. It has been tested that 70% percent ethanol concentrate will kill off 99.9% of germs or bacteria. One thing that causes hand sanitizers to be more effective than hand soap is the contact time it has with the skin.
Negative Properties of Ethanol as Disinfectants:
If the concentrate is under 60% it will not be as effective and maybe even just cause the bacteria to spread around more. Since ethanol kills germs by dehydration, it can sometimes be selective and therefore does not kill certain species of bacteria that actually resist ethanol (sterilizing selectivity). For example, bacterial spores are not typically affected by ethanol. Because of its selectivity, ethanol tends to work better on a pure mixture versus a mixed culture.
Other Substitutes for Ethanol
Bleach tends to work most effectively when killing bacteria. In a lab, it was shown that it worked more effectively on both the mixed and pure culture on E.Coli. It also appeared to act more quickly and be less selective. Hand soaps use an agent called triclosan, which actually proved to be very ineffective and had hardly any effect on killing E.Coli. Therefore, hand sanitizers might actually be a safer bet when washing hands because of the longer amount of contact time and the more direct dehydration of bacteria cells.
Works Cited "Comparison study on disinfectant efficiency of." University of Kansas . University of Kansas,
n.d. Web. 20 Feb. 2012. <www.cte.ku.edu/gallery/visibleknowledge/sturm/files/Student%20Work%20Documents/final%20Lab%20Project/group_b_report.pdf>.
Abstract: Efficacy of ethanol against E. Coli. Saif Ahmed and Emily Nordquist. The purpose of this lab was to test how effective ethanol is against bacteria, specifically Escherichia Coli. To test how effective ethanol is against E. Coli, solutions of chicken broth were mixed in different percentages of dilution. There were ten 200 mL beakers of solution total; two were 100% chicken broth, two were 80% chicken broth and 20% ethanol, two were 60% chicken broth and 40% ethanol, two were 40% chicken broth and 60% ethanol, and the final two were 20% chicken broth and 80% ethanol. After all of the solutions were made and after bacteria colonies were made in ten different agar plates, the two were mixed. To mix them, 1 mL of each solution was put into their respective agar plates. After a night of settling, results showed that there was no bacteria in any of the agar plates except for the two that did not have any ethanol. However, the lab did produce some unexpected results: at the bottom of almost every single solution mixture was an unknown white substance. However, in beakers 3 and 4 which had ethanol levels of 20%, the white substance was not present. According to biofuelguide.net, biodiesel is produced from alcohol (ethanol) and vegetable or animal oils/fats when the alcohol level is about 20%, which was the level of alcohol in beakers 3 and 4.
Keywords: Ethanol, chicken broth, E. Coli, agar plate, Petri dish,
Introduction:
Several hand sanitizer brands like Purell and Germ-X advertise for killing 99.99% of germs. The active ingredient in each of these products is ethyl alcohol and each one contains a certain percentage of ethanol. Hand sanitizers have increasingly been placed in schools throughout the United States to prevent the spread of sickness and germs amongst the student body. However, questions remain as to how effective the some shelf brand hand sanitizers are that have less that 60% concentration of ethanol (Franklin 2006). However, sources such as The University of Chicago Press’s Infection Control and Hospital Epidemiology’s article state that the percentage of ethanol must be at or above 50% for the product to be effective in killing viruses and bacteria (Fendler, Groziak 2002). Therefore, the question remains as to which percentage of ethanol truly kills off bacteria and what ethanol concentration should be required in hand sanitizers.
The University of Chicago tested the efficacy of Purell (62% ethanol) by applying different viruses to the product and using the process of plating to determine the efficacy of the hand sanitizer in killing bacteria. This lab was modeled after The University of Chicago’s test and determines the efficacy of varied ethanol percentages in killing E.Coli. By testing percentages of ethanol varying from 0% to 80% concentration combined with chicken broth, or a nutrient broth for the E.Coli, this lab observed the actual percentage of ethanol needed to kill off E.Coli, a very common bacteria. After letting the E.Coli grow in a separate nutrient broth for two days to feed and grow, it was then mixed with the ethanol-chicken broth solutions. The University of Chicago noted that the viruses were killed in approximately thirty seconds (Fendler, Groziak 2002). Therefore, the ethanol was expected to kill the E.Coli relatively quickly. Samples of each solution were taken and placed in an agar, a gelatinous substance, to begin the process of plating and counting.
Plates contain nutrient in the agar for bacteria to feed on and reveal single colonies of bacteria and can be “counted.” This process of counting showed how many colonies were left over from the effects of each solution of ethanol and chicken broth. Two controls were also used to compare how much bacteria was present in the room since the lab was not completely free of bacteria, or sterilized. The ineffectiveness or effectiveness of the different solutions of ethanol and chicken broth can be compared to the different percentages of ethanol in different hand sanitizers. For example, one type of Purell is 70% ethanol while Target Brand and Germ-X are closer to 62-65% ethanol. Therefore, the 80% ethanol solution is more comparable to Purell whereas the 60% ethanol is more comparable to Target Brand and Germ-X sanitizers.
Methods:
The first step of this lab is to measure out chicken broth and ethanol into different levels of concentration. With a total of ten beakers, 2 will be filled with 200 mL of chicken brother, 2 will be filled with 160 mL chicken broth and 40 mL of ethanol, 2 will be filled with 120 mL of chicken broth and 80 mL of ethanol, and so on. After all the different mixtures are made, 10 bacteria colonies will be made in Petri dishes using natural agar. After the colonies are made, the number of bacteria will need to be counted. Once all the bacteria in each dish is counted, mix the solutions with the bacteria colonies and let sit for a while. After at least a day of letting sit, return to the Petri dishes and count the number of bacteria in each solution. This will tell you which percentage of ethanol concentration is the most effective.
Results:
Table 1: Observations of solutions of Chicken Broth and Ethanol (No E. Coli)
Table 2: Chicken Broth + E. Coli + Ethanol (dishes 5-10 have too much solution)
The results from our experiment show that when the ethanol percentage is above 20%, E. Coli is eliminated.
Figure 1: Petri dish #1 – Little to no bacterial evidence
Figure 2: Petri dish #4 – bacteria colonies scattered across the dish
Discussion
The lab yielded unexpected results and refuted the hypothesis that the E.Coli would survive under a 60% concentration of ethanol alcohol. When referring to figures 13-23, it remains clear that bacteria were only present in Petri-dishes 1 & 2. In other words, bacteria only grew and survived in the controls which contained 100% chicken broth and 0% bacteria. This proves that the chicken broth did work as a sufficient nutrient broth for E.Coli. However, this also shows that E.Coli did not survive in 20% or higher concentrations of ethanol. This, however, does not allow one to conclude that 20% ethanol concentrations kills all bacteria due to the fact an unexpected reaction between the chicken broth ethanol took place and formed a white substance (Figures 18-19). This white substance appeared in all beakers besides beakers 3 & 4 (20% ethanol concentrations) and appeared more solid in beakers 5-10 and more liquid like in beakers 1 & 2. This provides evidence that perhaps a type of fat or oil settled to the bottom of beakers 1 & 2 due to natural separation, whereas in beakers 3-10 a reaction occurred between a type of oil or lipid with the ethanol. The reason that there was no substance present in beakers 3-4 is perhaps because the ethanol and chicken broth had fully reacted with each other at a 4-1 ratio (80% chicken broth, 20% ethanol) and no layers were able to form, where as beakers 5-10 did not have a perfect reaction ratio and therefore began to create layers.
This lab indicates that E.Coli grown in chicken broth will be killed in ethanol concentrations above 20%. It also shows that a more accurate test to determine at what concentration of ethanol E.Coli will be killed would be to test ethanol percentages between 0% and 20%. This lab also suggests that research should be done about the specific ingredients, primarily the oils and lipids in chicken broth and how they react with ethanol alcohol. Most likely, the ingredient will have a 4:1 reaction ratio due to the fact that no white substance was found in beakers 3 & 4. Another question to look at would be how well does chicken broth work to grow E.Coli versus a standard nutrient broth. One potential error that could have effect the lab results was also that each solution perhaps did not get an equal amount of E.Coli due to the fact the E.Coli was mixed in a liquid solution and did not guarantee equal distribution. The white substance formed could have also had an effect on killing E.Coli. Comparing these results to the varying percentages of ethanol in hand sanitizers; it appears as though when E.Coli is grown in circumstances similar to the chicken broth nutrient solution, it will be killed with 20% ethanol hand sanitizers. However, this lab does not guarantee that other types of bacteria will also follow these standards or guarantee that E.Coli not grown under circumstances like the chicken broth will follow these given standards.